The present invention relates generally to the field of analytical chemistry and, more specifically, to practical applications of compounds that support photo-induced electron transfer.
Partially reduced reactive oxygen species (ROS) are prevalent in living systems. For instance, such species are implicated in a variety of biological mechanisms, such as the by-products of reactions between oxidative enzymes and their substrates. Hydrogen peroxide (H2O2) is an archetypal ROS and, hence, it is an attractive candidate for chemical probes of biological processes and, in general, for analytical applications where H2O2 may be generated.
Accordingly, traditional probes are designed to be sensitive to reduction-oxidation (redox) activity that is directly or indirectly related to hydrogen peroxide. For instance, probes in this regard typically include fluorophores that, only when oxidized, are amenable to straightforward detection by fluorescence. Whilst structurally diverse small molecules are suitable for this purpose in various degrees, a number of challenges present themselves regardless of the molecular scaffold of the probes. These challenges include, for instance, poor water solubility or incompatibility with water; the presence of moieties on a probe that can undergo side reactions with thiols, such as in cellular contexts; a need for external activating enzymes; and lack of membrane permeability.
Further, many traditional probes undergo irreversible oxidation and, accordingly, they can be used only once. Hence, such probes are incapable of reversibly responding to oxidation and reduction events. See N. Soh et al. “Design and Development of a Fluorescent Probe for Monitoring Hydrogen Peroxide Using Photoinduced Electron Transfer,” Bioorganic & Medicinal Chemistry 13(4) (2005) 1131-1139; N. Soh et al. “Novel Fluorescent Probe for Detecting Hydroperoxides With Strong Emission in the Visible Range,” Bioorganic & Medicinal Chemistry 16(11) (2006) 2943-2946; and N. Soh et al. “Swallow-tailed Perylene Derivative: a New Tool for Fluorescent Imaging of Lipid Hydroperoxides,” Org. Biomol. Chem. 5 (2007) 3762-3768.
In general, fluorescent probes can operate via a range of energy transfer mechanisms. For instance, some probes are based upon irreversible processes utilizing Förster resonance energy transfer (FRET), also known as fluorescence resonance energy transfer, which is a mechanism describing energy transfer between two chromophores. See A. Albers et al. “A FRET-Based Approach to Ratiometric Fluorescence Detection of Hydrogen Peroxide,” J. Am. Chem. Soc. 128 (2006) 9640-9641. Other fluorescent probes operate via an internal charge transfer (ICT) mechanism triggered, for instance, by an irreversible oxidation of the probe upon exposure to hydrogen peroxide. See D. Srikun et al. “An ICT-Based Approach to Ratiometric Fluorescence Imaging of Hydrogen Peroxide Produced in Living Cells,” J. Am. Chem. Soc. 130 (2008) 4596-4597.
The inventors are aware of recent evidence of a fluorescent probe that incorporates a disulfide moiety and that is capable of achieving reversible fluorescence responses to hydrogen peroxide. See E. W. Miller et al. “A Fluorescent Sensor for Imaging Reversible Redox Cycles in Living Cells,” J. Am. Chem. Soc. 129 (2007) 3458-3459. The probe in this instance is based upon internal charge transfer occurring within a single chromophore, like some of the irreversible fluorescent probes discussed above, not by simple photoinduced electron-transfer (PET).
There remains a need for efficient fluorescent probes that overcome traditional challenges to such probes, that are capable of responding reversibly to redox events, and that eliminate the presence of or need for chromophores that are operative in probes based upon ICT.